CN109603195B - Animal tissue degreasing method and application thereof - Google Patents
Animal tissue degreasing method and application thereof Download PDFInfo
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- CN109603195B CN109603195B CN201811478365.XA CN201811478365A CN109603195B CN 109603195 B CN109603195 B CN 109603195B CN 201811478365 A CN201811478365 A CN 201811478365A CN 109603195 B CN109603195 B CN 109603195B
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
- B01D11/0203—Solvent extraction of solids with a supercritical fluid
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Abstract
The invention discloses a degreasing method of animal tissues and application thereof, comprising the following steps: (1) pretreatment: cleaning animal tissue, sterilizing, cutting into pieces, blocks or mashing into powder; (2) and (3) dehydrating: dehydrating animal tissue with ethanol solution; (3) and (3) extraction: putting the animal tissue into a closed extraction kettle for supercritical carbon dioxide degreasing; (4) separation: and (3) the fat and fat-soluble impurities enter the separation kettle along with the carbon dioxide fluid, the fat and fat-soluble impurities are separated from the carbon dioxide, the carbon dioxide participates in the next extraction cycle, the fat and fat-soluble impurities are removed in the separation kettle, and the carbon dioxide-containing carbon dioxide-based carbon dioxide composite material is obtained through at least two extraction cycles. After the animal tissue degreasing method adopts supercritical carbon dioxide extraction treatment, the degreasing rate of the animal tissue is more than 99 percent, and fat-soluble impurities in the animal tissue can be thoroughly removed; on the other hand, after the treatment of the supercritical carbon dioxide, the potential animal virus removal rate of the animal tissue reaches more than 99 percent.
Description
Technical Field
The invention relates to the technical field of degreasing, in particular to a degreasing method of animal tissues and application thereof.
Background
At present, degreasing methods for animal tissues include chemical immersion extraction and supercritical fluid extraction. The chemical soaking extraction mainly uses alkaline solution (such as sodium hydroxide aqueous solution, through saponification reaction with fat), organic reagent (such as acetone, petroleum ether, ethyl acetate and other organic reagents, through similar compatibility principle extraction) and the like. Chemical degreasing generally takes a long time, chemical reagents are used in large amounts, the environment is polluted, and the reagents are easy to remain in animal tissues.
The supercritical fluid extraction utilizes the characteristics of small viscosity, large density and the like of some substances (such as carbon dioxide, water and nitrous oxide) in a supercritical state, has the advantages of strong solubility, good diffusivity, easy control and the like, can realize the extraction and separation of fat by the supercritical fluid by controlling the temperature and the pressure, and can efficiently and thoroughly remove substances such as fat and the like in animal tissues. Meanwhile, the supercritical carbon dioxide extraction has the advantages of low supercritical temperature, cleanness, no residue, wide carbon dioxide source, low price, easy obtainment, safety, no toxicity, no combustion and the like, and has wide application prospect. However, supercritical fluid extraction has high requirements on equipment, and maintenance energy consumption and safety of supercritical conditions need to be considered.
Most of the commonly used animal tissue virus inactivation methods are strong acid and strong alkali solution treatment, ion ray irradiation treatment and the like, but the methods can cause chemical bond breakage on animal tissues to cause weakening of mechanical strength, and excessive treatment on the animal tissues can cause protein denaturation to influence the application effect of the material in vivo and the like.
Disclosure of Invention
Based on the problems, the invention aims to overcome the defects of the prior art and provide a method for degreasing animal tissues, wherein the degreasing rate can reach more than 99%, and meanwhile, potential viruses in the animal tissues can be removed, and the removal rate can reach more than 99%; meanwhile, the tissue degreased by the method has no chemical reagent residue, the mechanical strength of the animal tissue cannot be weakened, and protein denaturation in the animal tissue cannot be caused.
In order to achieve the purpose, the invention adopts the technical scheme that: a method of defatting animal tissue comprising the steps of:
(1) pretreatment: cleaning animal tissue, sterilizing, cutting into pieces, blocks or mashing into powder;
(2) and (3) dehydrating: dehydrating animal tissue with ethanol solution;
(3) and (3) extraction: putting the animal tissue into a closed extraction kettle for supercritical carbon dioxide degreasing;
(4) separation: and (3) the fat and fat-soluble impurities enter the separation kettle along with the carbon dioxide fluid, the fat and fat-soluble impurities are separated from the carbon dioxide, the carbon dioxide participates in the next extraction cycle, the fat and fat-soluble impurities are removed in the separation kettle, and the carbon dioxide-containing carbon dioxide-based carbon dioxide composite material is obtained through at least two extraction cycles. Wherein the animal tissue is derived from tissue or organ such as cortical bone, cancellous bone, cartilage, peritoneum, pericardium, lipoomentum, small intestine submucosa, bladder matrix, dermis, etc.
Preferably, the length and width of the sheet in the step (1) are 10-300 mm, and the volume of the block is 0.5-10.0 cubic centimeter.
Preferably, in the step (2), the animal tissue is dehydrated for 0.5 to 5.0 hours by using 75 to 100 percent ethanol solution.
Preferably, dynamic extraction degreasing is adopted in the step (3). Preferably, in the step (3), the carbon dioxide fluid is subjected to purification treatment before entering the extraction kettle and contacting with animal tissues, and the purification treatment is carried out through an ultrafiltration membrane with a certain ultrastructure.
Preferably, the degreasing temperature in the step (3) is 40-60 ℃, and the pressure is 20-40 MPa.
Preferably, the flow rate of the carbon dioxide in the step (3) is 18-20 Hz.
Preferably, the dynamic extraction degreasing time is 0.5-20.0 hours. Preferably, the volume of the extraction kettle is 0.5-150.0 liters, a stainless steel filter screen is arranged in the extraction kettle, and the filtering precision is 5-25 microns.
Preferably, when the supercritical carbon dioxide is used for degreasing in the step (3), an entrainer is added into the extraction kettle. It should be noted that the entrainer is used to mix with the extraction solvent (such as carbon dioxide), change the polarity of the solvent, improve the solute dissolving capacity of the solvent, significantly improve the extraction rate of the substances that are difficult to extract or have low extraction rate, or improve the separation effect.
Preferably, the entrainer is at least one of methanol, ethanol, acetone, ethyl acetate and phenethyl alcohol, and the mass concentration of the entrainer is 0.1-10%.
Preferably, the temperature of the separation kettle in the step (4) is 30-70 ℃.
In another aspect, the present invention provides the above degreasing method for removing or separating fat and fat-soluble substances from soft and hard tissues of animal origin.
The invention also provides a degreasing method for removing the fat and fat-soluble substances of the animal source material and removing or removing potential animal viruses.
In conclusion, the beneficial effects of the invention are as follows:
after the animal tissue degreasing method adopts supercritical carbon dioxide extraction treatment, the degreasing rate of the animal tissue is more than 99 percent, and fat-soluble impurities in the animal tissue can be thoroughly removed; on the other hand, after the treatment of the supercritical carbon dioxide, the potential animal virus removal rate of the animal tissue reaches more than 99 percent, and the animal virus can not be detected in the animal tissue material after the treatment of the method; the treatment method can also be used in combination with acid-base solution, and has better effect of removing animal viruses.
Detailed Description
The invention relates to a method for degreasing animal tissues by supercritical carbon dioxide extraction, which adopts unique process control, has the degreasing rate of over 99 percent, and can efficiently and quickly degrease the animal tissues, wherein the animal tissues comprise biomembranes such as bones, cartilages, pericardium and the like, dermis and the like. Meanwhile, the method can also be used for removing potential animal viruses in animal tissues, the removal rate of the animal viruses reaches more than 99 percent, and animal tissue materials cannot be detected to have the animal viruses after being treated by the method; meanwhile, the method can be used together with acid-base solution, and the treatment effect is better.
In some embodiments, the degreasing method of the present invention includes the process steps of pretreatment, dehydration, extraction, separation, and the like, and specifically includes the following steps:
a. pretreatment: cleaning and disinfecting animal tissues, and cutting the animal tissues into slices, blocks or smashing the animal tissues into powder, wherein the length and the width of each slice are 10-300 mm, and the volume of each block is 0.5-10.0 cubic centimeters;
b. and (3) dehydrating: dehydrating the animal tissue by using 75-100% ethanol solution for 0.5-5.0 hours;
c. and (3) extraction: after ethanol dehydration treatment, putting animal tissues into a closed extraction kettle for supercritical carbon dioxide degreasing, wherein the degreasing temperature is 40-60 ℃, and the pressure is 20-40 MPa; dynamically extracting and degreasing the blocky or flaky animal tissues, wherein the flow of carbon dioxide is 18-20 Hz, and the time for dynamically extracting and degreasing is 0.5-20.0 hours; entrainers are added in the supercritical carbon dioxide extraction process;
d. separation: and (3) allowing fat and fat-soluble impurities to enter a separation kettle along with the carbon dioxide fluid, adjusting the temperature to be 30-70 ℃, separating substances such as lipid from the carbon dioxide, allowing the carbon dioxide to participate in the next extraction cycle, and removing the substances such as fat in the separation kettle to finish degreasing.
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
Example 1
One embodiment of the method for defatting animal tissue of the present invention comprises the steps of:
(1) pretreatment: cleaning and sterilizing animal tissue cartilage, and cutting into slices with length and width of 10 mm × 300 mm;
(2) and (3) dehydrating: animal tissue is dehydrated for 0.5 hour by 75 percent ethanol solution;
(3) and (3) extraction: after ethanol dehydration treatment, animal tissues are put into a closed extraction kettle for supercritical carbon dioxide degreasing, wherein the degreasing temperature is 40 ℃, and the pressure is 20 MPa; dynamically extracting and degreasing the flaky animal tissues, wherein the flow of carbon dioxide is 20Hz, and the time for dynamically extracting and degreasing is 0.5 hour; entrainer methanol (mass concentration 0.1%) is added in the supercritical carbon dioxide extraction process;
(4) separation: and (3) allowing fat and fat-soluble impurities to enter a separation kettle along with the carbon dioxide fluid, adjusting the temperature to be 30 ℃, separating substances such as lipid from the carbon dioxide, allowing the carbon dioxide gas to participate in the second extraction cycle, and removing the substances such as fat in the separation kettle to obtain the lipid-soluble carbon dioxide.
Example 2
One embodiment of the method for defatting animal tissue of the present invention comprises the steps of:
(1) pretreatment: cleaning and sterilizing animal tissue pericardium, and cutting into blocks with volume of 3 cubic centimeters;
(2) and (3) dehydrating: animal tissue is dehydrated for 2 hours by 90 percent ethanol solution;
(3) and (3) extraction: after ethanol dehydration treatment, animal tissues are put into a closed extraction kettle for supercritical carbon dioxide degreasing, wherein the degreasing temperature is 50 ℃, and the pressure is 40 MPa; the massive animal tissues are degreased by dynamic extraction, the flow rate of carbon dioxide is 18Hz, and the time of the dynamic extraction degreasing is 7 hours; entrainers are added in the supercritical carbon dioxide extraction process, and are mixed solution (mass concentration is 5%) of ethanol and ethyl acetate, wherein the mass ratio of the ethanol to the ethyl acetate is 1: 5;
(4) separation: and (3) allowing fat and fat-soluble impurities to enter a separation kettle along with the carbon dioxide fluid, adjusting the temperature to 50 ℃, separating lipid and other substances from the carbon dioxide, allowing the carbon dioxide gas to participate in the next extraction cycle, and removing the fat and other substances in the separation kettle to obtain the lipid-soluble carbon dioxide.
Example 3
One embodiment of the method for defatting animal tissue of the present invention comprises the steps of:
(1) pretreatment: cleaning animal tissue dermis, sterilizing, and mashing into powder.
(2) And (3) dehydrating: animal tissues were dehydrated for 5.0 hours with 100% ethanol solution.
(3) And (3) extraction: after ethanol dehydration treatment, animal tissues are put into a closed extraction kettle for supercritical carbon dioxide degreasing, wherein the degreasing temperature is 60 ℃, and the pressure is 30 MPa; the powdery animal tissues are degreased by dynamic extraction, the flow rate of carbon dioxide is 19Hz, and the time of the dynamic extraction degreasing is 20.0 hours; entrainer acetone (with mass concentration of 10%) is added in the supercritical carbon dioxide extraction process;
(4) separation: and (3) allowing fat and fat-soluble impurities to enter a separation kettle along with the carbon dioxide fluid, adjusting the temperature to 70 ℃, separating lipid and other substances from the carbon dioxide, allowing the carbon dioxide gas to participate in the second extraction cycle, and removing the fat and other substances in the separation kettle to obtain the lipid-soluble carbon dioxide.
Example 4
One embodiment of the method for defatting animal tissue of the present invention comprises the steps of:
1) cleaning fresh pig heart envelope tissue, sterilizing, and cutting into slices with length and width of 150 mm × 150 mm;
2) and (3) dehydrating: dehydrating for 2 hours by using an absolute ethyl alcohol solution;
3) and (3) extraction: placing the pericardium tissue into an extraction kettle after dehydration treatment, and dynamically extracting for 8 hours at the temperature of 45 ℃, the pressure of 30MPa, the carbon dioxide flow of 18Hz and the entrainer of 5% ethanol by mass concentration;
4) separating out fat and other substances in the separation kettle at the temperature of 60 ℃, introducing carbon dioxide into the next extraction cycle, and performing supercritical fluid extraction to obtain defatted animal tissue.
Example 5 Effect test of degreasing method of the invention
(1) Fat content detection refers to the second acid hydrolysis method of GB 5009.6-2016.
Weighing the degreased pericardium of the embodiment 4, placing the degreased pericardium into a test tube, adding a hydrochloric acid solution, heating and hydrolyzing in a water bath kettle at 70-80 ℃ until the pericardium is completely digested, then adding an ethanol solution, cooling, transferring into a measuring cylinder with a plug, adding anhydrous ether for extraction, taking out an upper ether layer after extraction, placing the upper ether layer in a conical flask, heating to remove ether, and drying the residual solid to constant weight to obtain the fat content in the product.
The results show that the pericardium tissue defatting rate reaches 99.5%.
(2) Viral titer was tested using the cytopathic method (CPE).
The specific method comprises the following steps: weighing the core envelopes, adding corresponding indicator viruses (including Bovine Viral Diarrhea Virus (BVDV), porcine pseudorabies virus (PRV), encephalomyocarditis virus (EMCV) and Porcine Parvovirus (PPV)) respectively, culturing, treating by adopting the method of example 4, adding serum-free culture medium for grinding, performing sterile filtration on supernatant, observing the cytopathic condition of each gradient dilution by adopting a 96-well plate cytopathic Condition (CPE) method, and calculating the lgTCID50/0.1 ml of the 4 indicator viruses in the test group and the control group according to the Reed-Muench method.
CPE method: cells in logarithmic growth phase were seeded in 96-well plates (10 per well)4One), 5% CO2Culturing in an incubator at 37 ℃ for 24 hours to allow the cells to adhere to the wall and form a uniform monolayer; gradient dilution of Virus to 10-5～10-139 gradients of 100 microliters per well, one dilution per column, for 9 columns; adding culture solution into the rest 3 columns as blank control; 37 ℃ and 5% CO2Culturing for 10 days. The cytopathic condition was observed under a microscope every day, and each of the disease conditions was recorded according to the Reed-Muench formula:
TCID50 ═ CPE > 50% virus dilution + (> 50% percent-50)/(> 50% percent- < 50% percent) log 10;
Δ lgTCID50 represents an order of magnitude of viral reduction greater than 4 after treatment, indicating a removal rate of greater than 99.99%;
viral titers were calculated and the results expressed as infectious units per milliliter (IFU/ml).
The results are shown in Table 1, the virus removal rate of the method is more than 99%, and the variation of the log values of the 4 kinds of the indicated virus titers is more than 4logs, so that the method meets the requirements of relevant national regulations and standards.
TABLE 1 results after treatment of 4 indicator viruses by the method of the invention
| Indicative of virus | BVDV | PRV | EMCV | PPV |
| △lgTCID50 | ≥7.5 | ≥6.9 | ≥6.7 | ≥4.2 |
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (5)
1. A method for degreasing animal tissues, comprising the steps of:
(1) pretreatment: cleaning animal tissue, sterilizing, cutting into pieces, blocks or mashing into powder;
(2) and (3) dehydrating: dehydrating animal tissue with ethanol solution; in the step (2), 75-100% ethanol solution is adopted to dehydrate the animal tissue for 0.5-5.0 hours;
(3) and (3) extraction: putting the animal tissue into a closed extraction kettle for supercritical carbon dioxide degreasing; when the supercritical carbon dioxide is used for degreasing in the step (3), an entrainer is added into the extraction kettle, wherein the entrainer is ethanol; the mass concentration of the ethanol is 0.1-10%, and the step (3) adopts dynamic extraction degreasing; the degreasing temperature is 40-60 ℃, and the pressure is 20-40 Mpa; the dynamic extraction degreasing time is 0.5-20.0 hours;
(4) separation: and (3) the fat and fat-soluble impurities enter the separation kettle along with the carbon dioxide fluid, the fat and fat-soluble impurities are separated from the carbon dioxide, the carbon dioxide participates in the next extraction cycle, the fat and fat-soluble impurities are removed in the separation kettle, and the carbon dioxide-containing carbon dioxide-based carbon dioxide composite material is obtained through at least two extraction cycles.
2. The degreasing method according to claim 1, wherein the sheet-shaped material in step (1) has a length and width of 10 to 300 mm, and the bulk material has a volume of 0.5 to 10.0 cc.
3. The degreasing method according to claim 1, wherein the temperature of the separation kettle in the step (4) is 30-70 ℃.
4. The degreasing method according to claim 1, wherein the degreasing method is used for removing fat and fat-soluble substances from animal-derived soft and hard tissues.
5. The degreasing method of claim 1, wherein the degreasing method is used for removing fat and fat-soluble substances of animal-derived materials and removing potential animal viruses.
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| US3966981A (en) * | 1974-11-26 | 1976-06-29 | The United States Of America As Represented By The Secretary Of Agriculture | Process for removing residual solvents |
| CN101139543A (en) * | 2007-10-29 | 2008-03-12 | 中国农业大学 | Method for extracting sheep placental extract grease by over-critical carbon dioxide |
| CN101812087A (en) * | 2010-05-07 | 2010-08-25 | 青岛海恩赐生物工程有限公司 | Method for extracting high-purity phospholipid from sleeve-fish-processing waste |
| CN102771620A (en) * | 2012-07-14 | 2012-11-14 | 李世泰 | Method for producing hydrolyzed brain protein powder and cephalin by grease removal of supercritical carbon dioxide |
| CN106244740A (en) * | 2016-08-30 | 2016-12-21 | 张玉斌 | A kind of employing supercritical CO2abstraction technique carries out the method for Corii Sus domestica defat |
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| WO2015015631A1 (en) * | 2013-08-02 | 2015-02-05 | 株式会社日立製作所 | Solid/liquid separation apparatus, and method for same |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3966981A (en) * | 1974-11-26 | 1976-06-29 | The United States Of America As Represented By The Secretary Of Agriculture | Process for removing residual solvents |
| CN101139543A (en) * | 2007-10-29 | 2008-03-12 | 中国农业大学 | Method for extracting sheep placental extract grease by over-critical carbon dioxide |
| CN101812087A (en) * | 2010-05-07 | 2010-08-25 | 青岛海恩赐生物工程有限公司 | Method for extracting high-purity phospholipid from sleeve-fish-processing waste |
| CN102771620A (en) * | 2012-07-14 | 2012-11-14 | 李世泰 | Method for producing hydrolyzed brain protein powder and cephalin by grease removal of supercritical carbon dioxide |
| CN106244740A (en) * | 2016-08-30 | 2016-12-21 | 张玉斌 | A kind of employing supercritical CO2abstraction technique carries out the method for Corii Sus domestica defat |
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